Antibiotic ac-541 and production thereof



July 28,

2 Sheets-Sheet 1 Filed Aug. 27. 1969 NNm O v Ow Om OO 00 0w 000v (.LNBOHHd) HONVllIWSNVEIJ.

INVEN ORS WERNER KARL HAUgMANN VLADIMIR ZBINOVSKY ANTHONY JSEP SHAY y 2 1970 w. K. HAUSMANN ETAL. 3,522,349

ANTIBIOTIC AC-541 AND PRODUCTION THEREOF Filed Aug. 27, 1969 2 Sheets-Sheet z CPS ( 5 VALUES) PARTS PER MILLION I INVENTORS WERNER KARL HAUSMANN XllSNHlNl HAHN 13H VLAD'MIR ZBINOVSKY I ANTHONY JO P SHAY ATT NEY United States Patent Int. Cl. A61k 21/00 US. Cl. 424-116 1 Claim ABSTRACT OF THE DISCLOSURE A new antibiotic is produced, designated AC-541 by cultivating a new strain of Streptomyces hygroscopicus NRRL 3111. The new antibiotic is active against grampositive and gram-negative organisms and thus is useful in inhibiting the growth of such bacteria wherever they may be found.

This application is a continuation in-part of our copending application Ser. No. 359,193, filed Apr. 13, 1964, now abandoned.

This invention relates to a new antibiotic, to its production by fermentation, to methods for its recovery and concentration from crude solutions, to processes for its purification and to methods for the preparation of its salts.

The present invention includes within its scope the antibiotic in dilute forms, as crude concentrates, and in pure crystalline forms. These novel products are active against a variety of microorganisms including gram-positive and gram-negative bacteria. The eifects of the new antibiotic on specific microorganisms, together with its chemical and physical properties, differentiate it from previously described antibiotics.

The new antibiotic which we have designated AC-541 is formed during the cultivation under controlled conditions of a new strain of Streptomyces hygroscopicus. A viable culture of the new strain of S. hygroscopicus has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division, U. S. Department of Agriculture, Peoria, Ill., and has been added to its permanent collection. It is freely available in this repository under its Accession Number NRRL 3111.

The following is a general description of the 'organism based on the diagnostic characteristics observed. The un- 2 derscored descriptive colors are taken from the Color Harmony Manual, 3rd ed. (1948).

Amount of growth.Moderate on most media, light on Czapeks Solution Agar.

Aerial mycelium and/ or en masse spore colon-Aerial mycelium whitish becoming Fawn (3 ig or 4 ig) to Beaver (3 li or 4 li) to Beige (3 go) in sporulation zones. Spore masses developing hygroscopic areas on many media.

Soluble pigment.Absent on most media; yellowishbrown in light amounts on Tomato Paste Oatmeal agar.

Reverse color.-In cream to tan or light yellowish shades.

Miscellaneous physiological reactions.Nitrates reduced to nitrites in organic nitrate broth but not in inorganic nitrate broth; good gelatin liquefaction; no chromogenicity on peptone-iron agar. Carbon source utilization according to Pridham et al. [J. Bact., 56: 107-114 (1948)] as follows: Good to fair utilization of l-arabinose, d-fructose, lactose, d-mannitol, d-melibiose, d-raffinose, l-rhamnose, sucrose, dextrose, salicin and d-Xylose; poor to non-utilization of i-inositol, adonitol, dextran, d-melezitose and d-trehalose.

Morphology.Aerial mycelium giving rise to clusters of tightly spiralled and coiled spore chains. Spores mostly short cylindrical, 0.8-1.0].6 x 0.81.2,U.. Spores surface smooth as determined by electron microscopy.

When following Pridham et al. [A Guide for the Classification of Streptomycetes According to Selected Groups, Appl. Microbiol, 6:52-79 (1958)], the combination of tightly coiled and spiralled spore chains with gray-brown en-masse spore c'olor places the new isolate in the Grey series of the section spira. Of the species contained within this series, this new culture most closely corresponds to the concept of Streptomyces hygroscopicus. Comparison of the new culture with several NRRL,

ATCC and CBS reference strains of this species confirms the identification.

A critical examination of the cultural, physiological and morphological features of the organism was made when it'was grown on several media, including those recommended by Pridham et al. [A Selection of Media for Maintenance and Taxonomic Study of Streptomyces, Antibiotics Annual (1956-1957), pp. 947-953]. Detailed observations are recorded in Tables I, II, III and IV below. Underscored descriptive colors are from the Color Harmony Manual.

TABLE I.OULTURAL CHARACTERISTICS OF STREPTOMYCES HYGROSCOPICUS NRRL 3111 1 [Incubationz 14 Days; Temperature: 28 0.]

Amount of Soluble Medium Growth Aerial Mycelium and/or Spores Pigment Reverse Color Remarks Czapeks Solution Agar Light Aerial mycelium white becoming Fawn (4 None Cream (1% Ca) ig) in sporulating zones. Sporulation light.

Asparaglne Dextrose Agar.- M0de1ate. Aerial mycelium white becoming Fawn (4 .do.....

lg) to Beaver (4 li) in sporulating Zones.

Sporulation moderate.

Tomato Paste Agar do Aerial mycelium white, becoming Fawn (4 do lg) to Beaver (4 li) in sporulating Zones.

Pastel Yellow (1 db). Spore masses becoming dark and hygroscopic.

Light Wheat (2 ea). Spore masses strongly hygroscopic, be-

Sporulation moderate. coming black. Hickey and Tresners Agar do Aerial mycelium white, becoming Fawn (4 do.... Light Wheat (2 ea).... Spore masses beig) to Beaver (4 li) in sporulatmg Zones. coming black and Sporulation good. hygroscopic. Yeast Extract Agar do Aerial mycelium white, becoming Fawn (3 ...do..... Yellow Maple (3 ng).. Spore masses beig) to Beaver (3 li) in sporulation zones.

coming black and Sporulation good. hygroscopic. Oatflake Agar... .do Aerial mycelium white, becoming Fawn (4 .do..... Putty (1% ea) Hygroscopio character ig) to Beaver (4 li) in sporulation Zones. pronounced. Sporulation good. Carvajal s Oatmeal Agar do Aerial myceluim white, becoming Fawn (4 .do Citron (1 go) Spore masses beig) to Beaver (4 li) in sporulation Zones. coming black and Sporulation good. hygroscopic. Tomato Paste Oatmeal Agar do Aerial mycelium white, becoming Fawn (4 Yellow- Light Tan (3 go) Strongly hygroscopic.

ig) to Beaver (4 li) in sporulation zones. ish- Sporulation good. Pram,

lg Potato Dextrose Agar do Aerial mycelium white, becoming Fawn (4 None Light Wheat (2 ea).... Hygroscopic in zones ig) to Beaver (4 li) in sporulation Zones. of heaviest Sporulation moderate. sporulation. Bennett s Agar do.... Aerial mycelium white, becoming Fawn (4 .d0. Pastel Yellow (1 db). Hygroscopic in zones lg) to Beaver (4 11) in sporulation Zones. of heaviest Sporulation moderate. sporulation. Inorganic Salts-Starch Agar do... Aerial mycelium white, becoming Beige (3 do-...- Parchment (1% db)... Lightly hygroscopic.

go) to Fawn (3 lg) in sporulation zones.

Sporulation moderate.

3 4 TABLE II.Micromorphology of Streptomyces INOCULUM PREPARATION hygroscoplcus NRRL 3111 To prepare inoculum in shaker flasks, 100 ml. portions MediumCzapeks Solution Agar. of the following liquid medium in 500 ml. flasks are inoc- Aerial Mycelium and/ or Sporiferous Structures-Aerial ulated with an agar slant of the culture.

. h l n d gz ig i gt ggg g g f clusters of mg t y splra e Liquid medium: Grams per liter Spore S hapeSpores mostly short cylindrical. gfiizgz 3 Spore S1Z60.81.0p. 0.8-1.2,u. Molasses n Spore SurfaceSpore surface smooth, as determined by electron microscopy. 10 The flasks are incubated at about 28 C. on a recipro- TABLE III.-MISCELLANEOUS PHYSIOLOGICAL REACTIONS OF STREP- TOMYGES HYGROSCOPICUS NRRL 3111 [Incubation Temperature: 28 0.]

Incubation Amount of Medium Period Growth Physiological Reaction Organic Nitrate Broth 7 Days Heavy Nitrates reduced to nitrites.

o 14 Days do Do. Synthetic Nitrate Broth 7 Days Moderate- Nitrates not reduced to nitrites.

Do 14 Days. Heavy Do.

Moderate Partial liquefaction.

Gelatin 7 Days.

Do 14 Days do Complete liquefaction.

Iron-Peptone Agar- 24 Hours do Negative chromogenicity.

TABLE IV eating shaker and agitated vigorously for 24 to 72 hours, Carbon source utilization pattern of Streptomyces usually for 48 hours h roscopicus NRRL 3111 d TANK FERMENTATION [Incubationz 14 days; temperature: 28 0.]

Carbon Source; Utilization 1 For the production of the antibiotic in tank fermenta- Adonito] 0 tors the following medium is preferably used. l-Arab'nose 3 3O Dextra; 0 Fermentation medium: Grams per liter dlumctose 3 Corn steep liquor i-Inositol 1 Glucose Lactose 3 d-Mannit0] 3 ii 3 1 33: 322? Each tank is inoculated with from 0.1 to 10%, includ Rafl-mose 3 sive, of a culture broth fermented as described above for l Rhamnose 3 inoculum preparation. Aeration is supplied at the rate of Salicin 2 0 0.2-2.0 volumes, inclusive, of sterile air per volume of Sucrose u 3 broth per minute and the broth is agitated by an impeller driven at about 120-160 r.p.m. The temperature is maind'Trehalose 0 tained at 20-35 0., usually at 28 C. The fermentation $22122: may be continued for 24 to 240 hours, at which time the Negative control O mash 1s harvested 3: good utilization; 2:fair utilization; 1=poor utiliza- PURIFICATION PROCEDURE tion; O=no utilization.

It is to be understood that for the production of the t harvest the fermentation broth containing the antinew antibiotic the present invention is not limited to this blotle Preferably filtered at P 7 to remove the myeeliparticular organism or to organisms fully answering th D 1atomaceous earth or any of the conventional filtraabove growth and microscopic characteristics which are 9 alds y be to assist th fi trati n Whi h i 1 116 given for illustrative purposes. In fact, it is desired and Wlth Standard equipment Thereafter the antibiotic may intended to include the use of mutants produced from the be recovered f the filtrate PY adsorption on activated described organism by various means, such as X-radiation, Carbon, filtffltlofl and ellltloll With 80% methanel at P ultraviolet radiation, nitrogen mustard, phage exposure The ell-late 1S concentrated in Vaeue t0 epproxlmately V25 and the like. of the original mash volume and calcium ions are precipitated with sodium oxalate and removed by filtration. THE FERMENTATION PROCESS The filtrate is extracted with petroleum ether and then The cultivation of the new strain of S. hygroscopic-us passed through a column of a cation exchange resin at NRRL 3111 may take place in a variety of liquid culture neutral pH. The antibiotic is retained by the resin which media. Media which are useful for the production of the is thoroughly washed with water and methanol. The antinew antibiotic include an assimilable source of carbon biotic is then eluted by repeated suspension of the resin such as starch, sugar, molasses, glycerol, etc., an assimilain water of acid pH and filtration. The pooled filtrates are ble source of nitrogent such as protein, protein hydrolyadjusted to pH 5, filtered and lyophilized. This material sate, polypeptides, amino acids, corn steep liquor, etc., is chromatographed on a cation exchange resin at pH 7 and inorganic anions and cations, such as potassium, sodiby concentration gradient elution with an aqueous soluum, calcium, sulfate, phosphate, chloride, etc. Trace eletion of an inorganic salt, like Na SO NaCl, KCl, KBr, ments such as boron, molybdenum, copper, etc., are sup LiCl etc.

plied as needed in the form of impurities by other con- Two components, designated A aud B, are separated by stituents of the media. Aeration in tanks and bottles is selecting appropriate cuts and isolated by evaporation in provided by forcing sterile air through or onto the survacuo. Substance A is crystallized as the picrolonate. face of the fermenting medium. Further agitation is pro- Component B from the salt gradient column is devided in tanks, by a mechanical impeller. An antifoaming salted by chromatography on active carbon and lyophiagent, such as 1% octadecanol in lard oil may be added lized. In a solvent system consisting of phenol as needed. 75 parts), m-cresol (25 parts), acetic acid (4 parts), pyridine Carbon 1 35.68 Hydrogen 6.04 Nitrogen 18.58 Oxygen 24.35 Chlorine 12.31 Water of crystallization 3.83

The neutralization equavilent is 426 and a molecular weight of 458 is found by the differential vapor pressure method. The antibiotic contains no primary amino nitrogen and no methyl groups attached to an oxygen. The average percent of methyl groups attached to a nitrogen is 1.7 (as CH The pK values are at about 6.9 and 10.2 indicating a strongly basic character. The product has no melting point, but decomposes at about 200-215 C. It has an optical rotation of [a] 58 (c.=1.09 in water). The antibiotic exhibits no absorption at wavelengths of light from 220 my to 700 m,a.

An infrared absorption spectrum of component A as the hydrochloride salt in a KBr pellet is prepared in a standard manner. The compound exhibits absorption maxima in the infrared spectrum at the following wavelengths expressed in microns: 3.06, 5.84, 6.06, 6.25, 6.74, 7.24, 7.67, 8.43, 8.84, 9.36, 9.60, 10.56, 10.80. The infrared curve is shown in FIG. 1 of the accompanying drawmgs.

A proton magnetic resonance spectrum of component A as the hydrochloride salt is determined with a Varian A-60 spectrometer at 60 megacycles in the customary manner by dissolving in D (45 mg. in 0.35 ml.) containing tetramethylsilane as an internal standard. The compound has a characteristic resonance pattern with principal features occurring at the following frequencies expressed in 6 (delta) units (parts per million): 3.14, 3.55, 3.73, 3.85, 4.05, 4.16, 4.30, 4.49, 4.56, 4.65, 4.74, 4.89, 5.28, 5.62, 5.77, 5.80, 8.13 p.p.m. The resonance spectrum is shown in FIG. 2 of the accompanying drawings. Acid hydrolysis and paper chromatography of component A shows that ,B-lysine is absent.

In a solvent system consisting of 90% phenol (100 parts), m-cresol (25 parts), acetic acid (4 parts), pyridine (4 parts) and water (75 parts) the paper chromatographic Rf value of component A is about 0.58. The behavior in paper electrophoresis indicates that the compound is a strong base.

The hydrochloride is very soluble in water, somewhat less soluble in methanol, ethyleneglycol monomethyl ether, dimethylsulfoxide, dimethylformamide and practically insoluble in other common organic solvents such as the hydrocarbons. At 25 C. the antibiotic is most stable at pH 3-5, inactivation occurs above pH 7.

The compound dialyzes readily through cellophane. This new antibiotic is positive in the following color reactions: Tollens, Fehling, Biuret, permanganate, periodic acid. The Sakaguchi, ElsomMorgan, Molisch, maltol, ninhydrin, carbazole, anthrone and FeCl tests are negative.

Component A is clearly distinguished from other antibiotics by the characterization data given above and by its antimicrobial spectrum. The antimicrobial activity of component A is presented in the table below which shows the in vitro spectrum, that is, the zones of inhibition measured in millimeters from the edge of the agar wells to the outer limits of the inhibition zones at a concentration of 500 ,ag. per milliliter.

TABLE v In vitro antimicrobial spectrum of the A component by agar diffusion method Zone of inhibition Organism: at 500 ,ag/ml.

Bacillus cereus 7.6 Bacillus subtilis 11.6 Bacillus subtilis (pH 6 agar) 5.4

Bacillus subtilis (resistant to streptothricin) 9.1 Staphylococcus aureus (resistant to tetracyclines) 2.1 Staphylococcus aureus (strain Smith) 6.2 Staphylococcus aureus (resistant to erythromycin group) 2.0 Streptococcus pyogenes (NY-5) 3.3 Corynebacterium xerosis (NRRLB-1397) 6.3 Klebsiella pneumoniae (Friedlanders) 6.5 Klebsiclla pneumoniae 9.5 Klebsiella pneumoniae A (Strain AD) 11.6 Alcaligenes sp. (ATCC 10153) 6.8 Escherichia coli 6.3 Escherichia coli (resistant to chloramphenicol) 5.6 Salmonella gallinarum 7.6 Pseudomonas mucidolens (ATCC 4686) 10.7 Pseudomonas aeruginosa (ATCC 10145) 5.4 Proteus vulgaris (ATCC 9484) 5.5 Proteus mirabz'lis (ATCC 9921) 5.5

Component A is active against gram-negative and gram-positive microorganisms, such as Staphylococci, Pneumococci and Streptococci. The new antibiotic is thus potentially useful as a therapetic agent in treating bacterial infections in mammals caused by such microorganisms. The new antibiotic can be expected to be usefully employed for controlling such infections by topical application or parenteral administration.

The usefulness of the A component is demonstrated by its ability to control systemic lethal infections in mice. The new antibiotic shows high antibacterial in vivo activity in mice against Staphylococcus aureus, strain Smith, Streptococcus pyogenes C-203 and Escherichia call, when administered by single subcutaneous dose to groups of Carworth Farms CFl female mice, weight 18-20 grams, infected intraperitoneally with a lethal dose of these bacteria in a 10- to 10- trypticase soy broth (TSP) dilution of a five-hour TSP blood culture.

Table VI shows the in vivo antibacterial activity of Component A.

1 The antibiotic was administered in a single subcutaneous dose. N 3.1 051 surviving mice/number of treated mice. In controls, all injected mice e The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Inoculum preparation A typical medium used to grow the primary inoculum is prepared according to the following formula:

Grams Bactopeptone 5 Glucose 10 Molasses 20 Water to 1000' ml.

A yeast-malt agar slant of S. hydroscopicus NRRL 3111 is incubated for a week. At this time the spores and mycelium are transferred to two 500' ml. flasks which contain 100 ml. of the above medium. The flasks are placed on a reciprocating shaker and agitated vigorously for 48 hours, at 28 C. The flask inocula are transferred to 9 liter bottles, which contain 6 liters of the above liquid medium. These bottles are aerated for 24 hours to encourage further growth. At the end of this time the 9 liter bottles are used to seed fermentor tanks.

EXAMPLE 2 Fermentation A fermentation medium is prepared according to the following formula:

Grams Corn steep liquor 25 Glucose 30 (NH SO 3.3 CaCO 9 Water to 1000 ml.

sayed.

EXAMPLE 3 Isolation Four hundred and thirty liters of fermented mash at pH 7.5 is adjusted to pH 7.0 and diatomaceous earth is added in the proportion 3% weight per volume. The broth is filtered, the filter cake is washed with water and the pad is discarded. The water wash is pooled with the filtrate (total volume 400 liters) and 8.0 kg. of activated carbon is added. After filtration the antibiotic is eluted from the carbon by suspension in 200 liters of 80% methanol which is adjusted to pH 3 with HCl. The carbon is removed by filtration and discarded. The filtrate is concentrated to 19 liters (total solids 960 g.). Calcuim ions are precipitated with a saturated aqueous solution of sodium oxalate and removed by filtration. The filtrate is percolated through a column of ion exchange resin Amberlite IRC-50 (200-400 mesh) at pH 6.5. After washing thoroughly with water and methanol, the antibiotic is eluted from the resin by repeated suspension in water of pH 2.5 and filtration. The pooled filtrates (total volume 4.3 liters) are adjusted to pH with ion exchange resin Amberlite IR-45, filtered and lyophilized (total solids 208 g.). The product is chromatogranhed 8 on ion exchange resin Amberlite CG- (200400 mesh) by concentration gradient elution with an aqueous solution of KCl. The two components bands A and B are separated and individually isolated by evaporation. After extracting the activity from the dry product (28 g.) with methanol (275 ml.), antibiotic AC54l-A (2.3 g.) is crystallized as the picrolonate from aqueous acetone. The derivative is recrystallized from the same solvent (yield 1.9 g.). The crystalline picrolonate is then converted to the hydrochloride by passing an aqueous acetone solution through ion exchange resin Amberlite IR-45 in the hydrochloride form. The effluent is concentrated and lyophilized (yield 1 g.). The chemical analysis of this product as the hydrochloride and its other chemical, physical and biologcal properties have already been described.

What is claimed is:

1. A substance antibiotic AC-541-A effective in inhibiting the growth of gram-positive and gram-negative bacteria, the hydrochloride of which is characterized by the following properties:

(a) readily soluble in water, somewhat less soluble in lower alcohols, ethyleneglycol monomethyl ether, dimethylsulfoxide and dimethylformamide and insoluble in other common organic solvents,

(b) being basic and having pK values of 6.9 and 10.2,

(c) having an optical rotation [111 58 (c.=1.09

in water),

((1) an R value of 0.58 in a solvent system consisting of phenol parts), rn-cresol (25 parts), acetic acid (4 parts), pyridine (4 parts) and water (75 parts),

(e) containing the elements carbon, hydrogen, nitrogen, oxygen and chlorine in substantially the following average percentages by weight:

Carbon 35.68 Hydrogen 6.04 Nitrogen 18.58 Oxygen 24.35 Chlorine 12.31

Water of crystallization 3.83

(f) characteristic absorption in the infrared region of the spectrum as shown in FIG. 1, and (g) a proton resonance spectrum as shown in FIG. 2.

References Cited Miller: The Pfizer Handbook of Microbial Metabolites, McGraW-Hill Book Company, Inc., New York, N.Y., 1961, p. 591.

ALBERT T. MEYERS, Primary Examiner J. D. GOLDBERG, Assistant Examiner 

